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Although endometriosis is primarily benign, it has been identified as a risk factor for endometriosis-associated ovarian cancer (EAOC). Genetic alterations in ARID1A, PTEN, and PIK3CA have been reported in EAOC; however, an appropriate EAOC animal model has yet to be established. Therefore, the present study aimed to create an EAOC mouse model by transplanting uterine pieces from donor mice, in which Arid1a and/or Pten was conditionally knocked out (KO) in Pax8-expressing endometrial cells by the administration of doxycycline (DOX), onto the ovarian surface or peritoneum of recipient mice. Two weeks after transplantation, gene KO was induced by DOX and endometriotic lesions were thereafter removed. The induction of only Arid1a KO did not cause any histological changes in the endometriotic cysts of recipients. In contrast, the induction of only Pten KO evoked a stratified architecture and nuclear atypia in the epithelial lining of all endometriotic cysts, histologically corresponding to atypical endometriosis. The induction of Arid1a; Pten double-KO evoked papillary and cribriform structures with nuclear atypia in the lining of 42 and 50% of peritoneal and ovarian endometriotic cysts, respectively, which were histologically similar to EAOC. These results indicate that this mouse model is useful for investigating the mechanisms underlying the development of EAOC and the related microenvironment.

Although checkpoint inhibitors that block CTLA-4 and PD-1 have improved cancer immunotherapies, targeting additional checkpoint receptors may be required to broaden patient response to immunotherapy. PVRIG is a coinhibitory receptor of the DNAM/TIGIT/CD96 nectin family that binds to PVRL2. We report that antagonism of PVRIG and TIGIT, but not CD96, increased CD8+ T-cell cytokine production and cytotoxic activity. The inhibitory effect of PVRL2 was mediated by PVRIG and not TIGIT, demonstrating that the PVRIG–PVRL2 pathway is a nonredundant signaling node. A combination of PVRIG blockade with TIGIT or PD-1 blockade further increased T-cell activation. In human tumors, PVRIG expression on T cells was increased relative to normal tissue and trended with TIGIT and PD-1 expression. Tumor cells coexpressing PVR and PVRL2 were observed in multiple tumor types, with highest coexpression in endometrial cancers. Tumor cells expressing either PVR or PVRL2 were also present in numbers that varied with the cancer type, with ovarian cancers having the highest percentage of PVR−PVRL2+ tumor cells and colorectal cancers having the highest percentage of PVR+PVRL2− cells. To demonstrate a role of PVRIG and TIGIT on tumor-derived T cells, we examined the effect of PVRIG and TIGIT blockade on human tumor-infiltrating lymphocytes. For some donors, blockade of PVRIG increased T-cell function, an effect enhanced by combination with TIGIT or PD-1 blockade. In summary, we demonstrate that PVRIG and PVRL2 are expressed in human cancers and the PVRIG–PVRL2 and TIGIT–PVR pathways are nonredundant inhibitory signaling pathways.See related article on p. 244

Supplement 2

Figure captions

Supplement 1. Diagram describing the approach used for sampling of tissue blocks in Part 2 and Part 3 of the study.

Supplementary Figures and Tables

Supplement 3: Baseline characteristics for the 105 women that underwent extensive sampling of thefallopian tube.

Purpose:The current paradigm in the development of high-grade serous ovarian carcinoma (HGSC) proposes that the majority of HGSCs arise from precursor serous tubal intraepithelial carcinoma (STIC) lesions of the fallopian tube. Here we survey genome-wide methylation in HGSC precursor lesions to identify genomic regions that exhibit high-specificity differential hypermethylation for potential use as biomarkers for detecting STIC and HGSC at stages when curative intervention likely remains feasible.Experimental Design:We first identified quality control criteria for performing reliable methylomic analysis of DNA-limited tubal precursor lesions with the Illumina Infinium MethylationEPIC array. We then used this platform to compare genome-wide methylation among 12 STICs with paired adjacent-normal epithelia, one p53 signature lesion and two samples of concurrent HGSC. The resulting methylomic data were analyzed by unsupervised hierarchical clustering and multidimensional analysis. Regions of high-confidence STIC-specific differential hypermethylation were identified using selective bioinformatic criteria and compared with published MethylationEPIC data from 23 HGSC tumors and 11 healthy fallopian tube mucosae.Results:Unsupervised analysis showed that STICs largely clustered with HGSCs, but were clearly distinct from adjacent-normal fallopian tube epithelia. Forty-two genomic regions exhibited high-confidence STIC-specific differential hypermethylation, of which 17 (40.5%) directly overlapped with HGSC-specific differentially methylated regions. Methylation at these shared loci was able to completely distinguish STIC and HGSC samples from normal and adjacent-normal specimens.Conclusions:Our results suggest that most STICs are epigenetically similar to HGSCs and share regions of differential hypermethylation that warrant further evaluation for potential use as biomarkers for early detection of ovarian HGSC.See related commentary by Ishak and De Carvalho, p. 6083

Elucidating the earliest pathogenic steps in cancer development is fundamental to improving its early detection and prevention. Ovarian high-grade serous carcinoma (HGSC), a highly aggressive cancer, mostly originates from the fallopian tube epithelium through a precursor stage, serous tubal intraepithelial carcinoma (STIC). In this study, we performed spatial transcriptomic analysis to compare STICs, carcinoma, and their matched normal fallopian tube epithelium. Several differentially expressed genes in STICs and carcinomas were involved in cancer metabolism and detected in a larger independent transcriptomic dataset of ovarian HGSCs. Among these, insulin-like growth factor binding protein-2 (IGFBP2) was found to undergo DNA hypomethylation and to be increased at the protein level in STICs. Pyrosequencing revealed an association of IGFBP2 expression with the methylation state of its proximal enhancer, and 5-azacytidine treatment increased IGFBP2 expression. In postmenopausal fallopian tubes, where most STICs are detected, IGFBP2 immunoreactivity was detected in all 38 proliferatively active STICs but was undetectable in morphologically normal tubal epithelia, including those with TP53 mutations. In premenopausal fallopian tubes, IGFBP2 expression was limited to the secretory epithelium at the proliferative phase, and estradiol treatment increased IGFBP2 expression levels. IGFBP2 knockdown suppressed the growth of IGFBP2-expressing tubal epithelial cells via inactivation of the AKT pathway. Taken together, demethylation of the proximal enhancer of IGFBP2 drives tumor development by maintaining the increased IGFBP2 required for proliferation in an otherwise estrogen-deprived, proliferation-quiescent, and postmenopausal tubal microenvironment.Significance:Molecular studies of the earliest precursor lesions of ovarian cancer reveal a role of IGFBP2 in propelling tumor initiation, providing new insights into ovarian cancer development.

Supplementary Table 2 from Gene Expression Signatures Differentiate Ovarian/Peritoneal Serous Carcinoma from Diffuse Malignant Peritoneal Mesothelioma

Supplementary Figures S1-S5

Supplemental Materials, Supplemental Tables S1, S2,and S5, Supplemental Figures S1-S6

Purpose: Ovarian/primary peritoneal serous carcinoma (OC/PPC) and diffuse peritoneal malignant mesothelioma (DMPM) are highly aggressive tumors that are closely related morphologically and histogenetically. It remains unclear whether both tumors are molecularly distinct neoplasms. The current study compared global gene expression patterns in OC/PPC and DMPM.Experimental Design: Ten OC/PPC and five DMPM effusions were analyzed for gene expression profiles using the Affymetrix U133 Plus 2 arrays and the dCHIP analysis program. Differentially expressed candidate genes were validated using quantitative real-time PCR and immunohistochemistry.Results: Unsupervised hierarchical clustering using all 54,675 genes in the array classified the samples into two groups: DMPM specimens versus OC/PPC specimens. A total of 189 genes that were differentially expressed in these two groups were selected based on statistical significance. Genes overexpressed in DMPM (n = 68) included calretinin, vitronectin, claudin 15, α4 laminin, hyaluronan synthase 1, cadherin 11, RAB7, v-maf, and the epidermal growth factor–containing fibulin-like extracellular matrix protein 1. Genes overexpressed in OC/PPC (n = 121) included insulin-like growth factor II (IGF-II); IGF-II binding protein 3; cyclin E1; folate receptors 1 and 3; RAB25; MUC4; endothelin-1; CD24; kallikreins 6, 7, and 8; claudins 3, 4, and 6; Notch3; and MMP-7. Quantitative real-time PCR validated the differential expression of 13 genes, and immunohistochemistry confirmed the differences for four gene products.Conclusions: Expression profiling separates OC/PPC from DMPM and identifies a number of genes that are differentially expressed in these tumors. The molecular signatures unique to OC/PPC and DMPM should provide a molecular basis to study both tumors and new potential markers for facilitating their differential diagnosis.

Supplementary Tables S1-S8

Supplementary Figures

The evolution of chemoresistance is a fundamental characteristic of cancer that ultimately hampers its clinical management. However, it may be possible to improve patient outcomes significantly by a better understanding of resistance mechanisms, which cancers rely upon during the evolution to an untreatable state. Here we report an essential role of the stem cell reprogramming factor, PBX1, in mediating chemoresistance in ovarian carcinomas. In the clinical setting, high levels of PBX1 expression correlated with shorter survival in post-chemotherapy ovarian cancer patients. In tumor cells with low endogenous levels of PBX1, its enforced expression promoted cancer stem cell-like phenotypes, including most notably an increase in resistance to platinum-based therapy used most commonly for treating this disease. Conversely, silencing PBX1 in platinum-resistant cells that overexpressed PBX1 sensitized them to platinum treatment and reduced their stem-like properties. An analysis of published genome-wide chromatin immunoprecipitation data indicated that PBX1 binds directly to promoters of genes involved in stem cell maintenance and the response to tissue injury. We confirmed direct regulation of one of these genes, STAT3, demonstrating that the PBX1 binding motif at its promoter acted to positively regulate STAT3 transcription. We further demonstrated that a STAT3/JAK2 inhibitor could potently sensitize platinum-resistant cells to carboplatin and suppress their growth in vivo. Our findings offer a mechanistic rationale to target the PBX1/STAT3 axis to antagonize a key mechanism of chemoresistance in ovarian cancers and possibly other human cancers. Cancer Res; 76(21); 6351–61. ©2016 AACR.

Supplementary Tables 1-3, Supplementary Figures 1-7. Supplementary Table 1: Oligonucleotide primers used for qRT-PCR analysis. Supplementary Table 2: GSEA enrichment of KEGG functional pathways in lovastatin-regulated genes. Supplementary Table 3: Lovastatin affects expression of genes in glycolysis/gluconeogenesis pathway. Supplementary Figure 1: Daily administration of lovastatin prevents tumor growth in mogp-TAg transgenic mice. Supplementary Figure 2: Body weight and serum levels of cholesterol and triglyceride in mogp-TAg, SKOV3-IP, and OVCAR5 mouse tumor models. Supplementary Figure 3: Atorvastatin treatment delays growth of ovarian tumor xenografts. Supplementary Figure 4: Statin treatment increases transcript levels of LC3A and LC3B. Supplementary Figure 5: Scheme of the mevalonate pathway. Supplementary Figure 6: Applying GGPP or FPP as a single agent does not affect ovarian cancer cell proliferation. Supplementary Figure 7: Real-time qRT-PCR confirms knockdown efficiency of each siRNA.

Differentially expressed genes between different comparisons

Gene promoters identified by PBX1 ChIP-on-chip.

Supplementary Table 1 from Gene Expression Signatures Differentiate Ovarian/Peritoneal Serous Carcinoma from Diffuse Malignant Peritoneal Mesothelioma

Including Supplementary Figures and Methods

Purpose:High-grade serous ovarian carcinoma (HGSOC) typically remains undiagnosed until advanced stages when peritoneal dissemination has already occurred. Here, we sought to identify HGSOC-specific alterations in DNA methylation and assess their potential to provide sensitive and specific detection of HGSOC at its earliest stages.Experimental Design:MethylationEPIC genome-wide methylation analysis was performed on a discovery cohort comprising 23 HGSOC, 37 non-HGSOC malignant, and 36 histologically unremarkable gynecologic tissue samples. The resulting data were processed using selective bioinformatic criteria to identify regions of high-confidence HGSOC-specific differential methylation. Quantitative methylation-specific real-time PCR (qMSP) assays were then developed for 8 of the top-performing regions and analytically validated in a cohort of 90 tissue samples. Lastly, qMSP assays were used to assess and compare methylation in 30 laser-capture microdissected (LCM) fallopian tube epithelia samples obtained from cancer-free and serous tubal intraepithelial carcinoma (STIC) positive women.Results:Bioinformatic selection identified 91 regions of robust, HGSOC-specific hypermethylation, 23 of which exhibited an area under the receiver-operator curve (AUC) value ≥ 0.9 in the discovery cohort. Seven of 8 top-performing regions demonstrated AUC values between 0.838 and 0.968 when analytically validated by qMSP in a 90-patient cohort. A panel of the 3 top-performing genes (c17orf64, IRX2, and TUBB6) was able to perfectly discriminate HGSOC (AUC 1.0). Hypermethylation within these loci was found exclusively in LCM fallopian tube epithelia from women with STIC lesions, but not in cancer-free fallopian tubes.Conclusions:A panel of methylation biomarkers can be used to accurately identify HGSOC, even at precursor stages of the disease.

Supplementary Data from DNA Copy Numbers Profiles in Affinity-Purified Ovarian Clear Cell Carcinoma

In vivo phosphoproteome

Supplementary Tables S1-S3

Enriched IPA pathways inferred from PBX1-ChIP target genes.

Purpose: Statins are among the most frequently prescribed drugs because of their efficacy and low toxicity in treating hypercholesterolemia. Recently, statins have been reported to inhibit the proliferative activity of cancer cells, especially those with TP53 mutations. Because TP53 mutations occur in almost all ovarian high-grade serous carcinoma (HGSC), we determined whether statins suppressed tumor growth in animal models of ovarian cancer.Experimental Design: Two ovarian cancer mouse models were used. The first one was a genetically engineered model, mogp-TAg, in which the promoter of oviduct glycoprotein-1 was used to drive the expression of SV40 T-antigen in gynecologic tissues. These mice spontaneously developed serous tubal intraepithelial carcinomas (STICs), which are known as ovarian cancer precursor lesions. The second model was a xenograft tumor model in which human ovarian cancer cells were inoculated into immunocompromised mice. Mice in both models were treated with lovastatin, and effects on tumor growth were monitored. The molecular mechanisms underlying the antitumor effects of lovastatin were also investigated.Results: Lovastatin significantly reduced the development of STICs in mogp-TAg mice and inhibited ovarian tumor growth in the mouse xenograft model. Knockdown of prenylation enzymes in the mevalonate pathway recapitulated the lovastatin-induced antiproliferative phenotype. Transcriptome analysis indicated that lovastatin affected the expression of genes associated with DNA replication, Rho/PLC signaling, glycolysis, and cholesterol biosynthesis pathways, suggesting that statins have pleiotropic effects on tumor cells.Conclusions: The above results suggest that repurposing statin drugs for ovarian cancer may provide a promising strategy to prevent and manage this devastating disease. Clin Cancer Res; 21(20); 4652–62. ©2015 AACR.

Supplementary Figures and Methods

Supplementary Figure 3 from NAC-1 Controls Cell Growth and Survival by Repressing Transcription of Gadd45GIP1, a Candidate Tumor Suppressor

PDF file - 76K, Supporting data of quantitative binding assay

PDF file - 84K

PDF file - 147K, Effects of inactivating NAC1 in HeLa N130 tTA cell line

PDF file - 60K, Silencing of NAC1 in HeLa cell line

PDF file - 100K, Time lapse frames of shRNA treated HeLa cells

Cancer mortality and morbidity are primarily related to recurrent tumors, and characterization of recurrence-associated genes should illuminate fundamental properties of tumor progression and provide new therapeutic targets. We have previously identified NAC-1, a member of the BTB/POZ gene family and a transcription repressor, as a gene associated with recurrent ovarian carcinomas after chemotherapy. We further showed that homodimerization of NAC-1 proteins is essential for tumor growth and survival. In this study, we applied serial analysis of gene expression and identified growth arrest and DNA-damage–inducible 45-γ interacting protein (Gadd45GIP1) as one of the downstream genes negatively regulated by NAC-1. NAC-1 knockdown in both SKOV3 and HeLa cells that expressed abundant endogenous NAC-1 induced Gadd45GIP1 expression transcriptionally; on the other hand, engineered expression of NAC-1 in NAC-1–negative RK3E and HEK293 cells suppressed endogenous Gadd45GIP1 expression. In NAC-1–expressing tumor cells, induction of dominant negative NAC-1 conferred a growth-inhibitory effect that can be partially reversed by Gadd45GIP1 knockdown. Induced Gadd45GIP1 expression resulted in growth arrest in SKOV3 and HeLa cells both in vitro and in vivo. In summary, NAC-1 contributes to tumor growth and survival by at least inhibiting Gadd45GIP1 expression, which has a tumor suppressor effect in cancer cells. [Cancer Res 2007;67(17):8058–64]

Supplementary Table 1, Figures 1-7 from Jagged-1 and Notch3 Juxtacrine Loop Regulates Ovarian Tumor Growth and Adhesion

Rsf-1 interacts with human sucrose nonfermenting protein 2 homologue (hSNF2H) to form a chromatin remodeling complex that participates in several biological processes. We have previously shown that Rsf-1 gene amplification was associated with the most aggressive type of ovarian cancer and cancer cells with Rsf-1 overexpression depended on Rsf-1 to survive. In this report, we determine if formation of the Rsf-1/hSNF2H complex could be one of the mechanisms contributing to tumor cell survival and growth in ovarian carcinomas. Based on immunohistochemistry, we found that Rsf-1 and hSNF2H were co-upregulated in ovarian cancer tissues. Ectopic expression of Rsf-1 in SKOV3 ovarian cancer cells with undetectable endogenous Rsf-1 expression enhanced hSNF2H protein levels and promoted SKOV3 tumor growth in a mouse xenograft model. Our studies also indicated that induction of Rsf-1 expression affected the molecular partnership of hSNF2H and translocated hSNF2H into nuclei where it colocalized with Rsf-1. Furthermore, analysis of Rsf-1 deletion mutants showed that the Rsf-D4 fragment contained the hSNF2H binding site based on coimmunoprecipitation and in vitro competition assays. As compared with other truncated mutants, expression of Rsf-D4 resulted in remarkable growth inhibition in ovarian cancer cells with Rsf-1 gene amplification and overexpression, but not in those without detectable Rsf-1 expression. The above findings suggest that interaction between Rsf-1 and hSNF2H may define a survival signal in those tumors overexpressing Rsf-1. [Cancer Res 2008;68(11):4050–7]

NOTCH3 gene amplification plays an important role in the progression of many ovarian and breast cancers, but the targets of NOTCH3 signaling are unclear. Here, we report the use of an integrated systems biology approach to identify direct target genes for NOTCH3. Transcriptome analysis showed that suppression of NOTCH signaling in ovarian and breast cancer cells led to downregulation of genes in pathways involved in cell-cycle regulation and nucleotide metabolism. Chromatin immunoprecipitation (ChIP)-on-chip analysis defined promoter target sequences, including a new CSL binding motif (N1) in addition to the canonical CSL binding motif, that were occupied by the NOTCH3/CSL transcription complex. Integration of transcriptome and ChIP-on-chip data showed that the ChIP target genes overlapped significantly with the NOTCH-regulated transcriptome in ovarian cancer cells. From the set of genes identified, we showed that the mitotic apparatus organizing protein DLGAP5 (HURP/DLG7) was a critical target. Both the N1 motif and the canonical CSL binding motif were essential to activate DLGAP5 transcription. DLGAP5 silencing in cancer cells suppressed tumorigenicity and inhibited cellular proliferation by arresting the cell cycle at the G2–M phase. In contrast, enforced expression of DLGAP5 partially counteracted the growth inhibitory effects of a pharmacologic or RNA interference–mediated NOTCH inhibition in cancer cells. Our findings define direct target genes of NOTCH3 and highlight the role of DLGAP5 in mediating the function of NOTCH3. Cancer Res; 72(9); 2294–303. ©2012 AACR.

Notch3 gene amplification and pathway activation have been reported in ovarian serous carcinoma. However, the primary Notch3 ligand that initiates signal transduction in ovarian cancer remains unclear. In this report, we identify Jagged-1 as the highest expressed Notch ligand in ovarian tumor cells as well as in peritoneal mesothelial cells that are in direct contact with disseminated ovarian cancer cells. Cell-cell adhesion and cellular proliferation were reduced in Notch3-expressing ovarian cancer cells that were cocultured with Jagged-1 knockdown mesothelial and tumor feeder cells. Interaction of Notch3-expressing ovarian cancer cells with Jagged-1–expressing feeder cells activated the promoter activity of candidate Notch3 target genes, and this activity was attenuated by Notch3 siRNA. Constitutive expression of the Notch3 intracellular domain significantly suppressed the Jagged-1 shRNA–mediated growth inhibitory effect. In Notch3-expressing ovarian cancer cells, Jagged-1–stimulating peptides enhanced cellular proliferation, which was suppressed by γ-secretase inhibitor and Notch3 siRNA. Taken together, our results show that Jagged-1 is the primary Notch3 ligand in ovarian carcinoma and Jagged-1/Notch3 interaction constitutes a juxtacrine loop promoting proliferation and dissemination of ovarian cancer cells within the intraperitoneal cavity. [Cancer Res 2008;68(14):5716–23]

Supplementary Figures 1-6 from Functional Genomic Analysis Identified Epidermal Growth Factor Receptor Activation as the Most Common Genetic Event in Oral Squamous Cell Carcinoma

A 250K single-nucleotide polymorphism array was used to study subchromosomal alterations in oral squamous cell carcinoma (OSCC). The most frequent amplification was found at 7p11.2 in 9 of 29 (31%) oral cancer patients. Minimal genomic mapping verified a unique amplicon spanning from 54.6 to 55.3 Mb on chromosome 7, which contains SEC61G and epidermal growth factor receptor (EGFR). Results from fluorescence in situ hybridization, transcriptome, and immunohistochemistry analyses indicated that the expression level of EGFR, but not of SEC61G, was up-regulated and tightly correlated with DNA copy number in 7p11.2 amplified tumors. Among the members of the erbB family, EGFR (HER1) was found to be the most frequently amplified and highly expressed gene in both human and mouse oral tumors (P < 0.01). Genes for downstream effectors of EGFR, including KRAS, mitogen-activated protein kinase 1, and CCND1, were also found amplified or mutated, which resulted in activation of EGFR signaling in 55% of OSCC patients. Head and neck squamous cancer cells with different EGFR expression levels showed differential sensitivity to antitumor effects of AG1478, a potent EGFR inhibitor. AG1478-induced EGFR inactivation significantly suppressed tumor development and progression in a mouse oral cancer model. Our data suggest that EGFR signaling is important in oral cancer development and that anti-EGFR therapy would benefit patients who carry the 7p11.2 amplicon in their tumors. [Cancer Res 2009;69(6):2568–76]

PDF file - 68K, Domain mapping of NAC1 binding region

Supplementary Figure Legends 1-6 from Functional Genomic Analysis Identified Epidermal Growth Factor Receptor Activation as the Most Common Genetic Event in Oral Squamous Cell Carcinoma

Supplementary Figures 1-2 from The Roles of Human Sucrose Nonfermenting Protein 2 Homologue in the Tumor-Promoting Functions of Rsf-1

Gene amplification is one of the common mechanisms that activate oncogenes. In this study, we used single nucleotide polymorphism array to analyze genome-wide DNA copy number alterations in 31 high-grade ovarian serous carcinomas, the most lethal gynecologic neoplastic disease in women. We identified an amplicon at 19p13.12 in 6 of 31 (19.5%) ovarian high-grade serous carcinomas. This amplification was validated by digital karyotyping, quantitative real-time PCR, and dual-color fluorescence in situ hybridization (FISH) analysis. Comprehensive mRNA expression analysis of all 34 genes within the minimal amplicon identified Notch3 as the gene that showed most significant overexpression in amplified tumors compared with nonamplified tumors. Furthermore, Notch3 DNA copy number is positively correlated with Notch3 protein expression based on parallel immunohistochemistry and FISH studies in 111 high-grade tumors. Inactivation of Notch3 by both γ-secretase inhibitor and Notch3-specific small interfering RNA suppressed cell proliferation and induced apoptosis in the cell lines that overexpressed Notch3 but not in those with minimal amount of Notch3 expression. These results indicate that Notch3 is required for proliferation and survival of Notch3-amplified tumors and inactivation of Notch3 can be a potential therapeutic approach for ovarian carcinomas. (Cancer Res 2006; 66(12): 6312-8)

Supplementary Figures, Tables and Methods from Analysis of DNA Copy Number Alterations in Ovarian Serous Tumors Identifies New Molecular Genetic Changes in Low-Grade and High-Grade Carcinomas

PDF file - 63K, mOSE cells of NAC1 knockout mouse